Skin Color Control of the Red Sea Bream (Pagrus major)

Lebensm.-Wiss. u.-Technol., 31, 27–32 (1998)

Skin Color Control of the Red Sea Bream (Pagrus major) Mao Qun Lin, Hideki Ushio*, Toshiaki Ohshima, Hideaki Yamanaka and Chiaki Koizumi Department of Food Science and Technology, Tokyo University of Fisheries, Tokyo 108 (Japan) (Received March 7, 1997; accepted May 6, 1997) The effect of different concentration of K + and Na + on the skin colour of red sea bream and on melanosome movements in melanophores was investigated by using a video microscope system and subsequent image analysis. Soaking in artificial sea water containing 470 mmol/L NaCl, 10 mmol/L KCl, 10 mmol/L CaCl2, and 50 mmol/L MgCl2 failed to prevent skin darkening, while solutions containing over 300 mmol/L KCl instead of the corresponding amount of NaCl induced aggregation of melanosomes, effectively preventing skin darkening. However, soaking in a solution containing 480 mmol/L KCl diminished the red colour of the fish skin. It is concluded that the solution containing 300 mmol/L KCl is useful to maintain skin colour of red sea bream and improve its commercial value.

©1998 Academic Press Limited Keywords: fish; melanophore; erythrophore; red sea bream

Introduction Since a vivid colour of cultured red skinned fish, such as red sea bream (Pagrus major), has a high market value, many investigators have improved the skin colour by dietary control, such as feeding astaxanthin-enriched diet (1, 2). The resulting skin colour of the fish is very vivid in the living state. However, immediately after killing the fish for consumption, the colour tone quickly becomes dark, reducing the commercial value of the fish. It is generally accepted that melanophores play an important role in the rapid colour change of certain fish (3). Functions of the melanophore are mainly controlled by hormones or through autonomic neurons (4). The major hormones that regulate the chromatosome movements on the fish skin are the melanophorestimulating hormones and melanin-concentrating hormones from the intermediate lobe of the pituitary (3, 5, 6). Many physiological and pharmacological studies have revealed that rapid chromatosome movements (dispersion and aggregation) in melanophores are also controlled via autonomic neurons (3, 7–11). Therefore, the rapid skin colour change after killing of cultured sea bream is thought to be mainly due to the rapid dispersion of chromatosomes in melanophores elicited by handling and killing stresses. Potassium ions are also known to act on the termini of neurons to release stimulants such as noradrenaline and to induce aggregation of chromatosomes in melanophores (12–16). As well as melanophores there are other chromatophores, such as xanthophores and erythrophores, in *To whom correspondence should be addressed.

red-skinned fish. Since the colouring matter extracted from red fish, such as red sea bream, is almost entirely astaxanthin diester (17) it is assumed that erythrophores mainly contribute the red colour of the fish skin. Erythrophores of some fish species are able to move in response to stimulation (18–21). Smith and Smith (18) reported that potassium ions induced aggregation of chromatosomes in erythrophores of squirrel fish. In general, the rates of translocation of chromatosomes in erythrophores and xanthophores are lower than those of melanophores (8). It is possible that some physiological conditions might induce dispersion of chromatosome in erythrophores and aggregation in melanophores, preventing fish skins from darkening while maintaining the red colour. The goal of this study was to develop a method to regulate chromatosome movements in melanophores and erythrophores using selected concentrations of cations, such as Na + and K + , to express the vivid colour of red fish skin.

Table 1 Cation concentration (mmol/L) of experimental solutions Experimental solutions Cations Na+ K+ Ca2+ Mg2+

10

K+

470 10 10 50

50 K+

100 K+

300 K+

430 50 10 50

380 100 10 50

180 300 10 50

480 K+ 0 480 10 50

Chloride ion was used as a counter ion against individual cations.

0023-6438/98/010027 + 06 $25.00/0/fs970280

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©1998 Academic Press Limited

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Table 2

Macro program used in image analyses

melanophores during the initial 60 min immediately after spiking. A solution containing only 100 mmol/L KCl was also used in addition to the solutions listed in Table 1. These experiments were carried out at a room temperature (20 °C) because it was very hard to set the temperature of fish skin to 0 °C on the video microscope system. The image data acquired on a floppy diskette were converted into Macintosh format through SC-MA V.1.0 software (Fujix, Tokyo, Japan) and analysed in a personal computer Macintosh LC575 (Apple, U.S.A.)

begin Duplicate(‘Processed Window’); ApplyLUT; filter(‘smooth’); ReduceNoise; filter(‘sharpen’); SetThreshold(165a); MakeBinary; SetScale(1,‘pixel’,1); ResetCounter; SetParticleSize(5b,99999); LabelParticles(false); IncludeInteriorHoles(true); SetOptions(‘Area’); AnalyzeParticles; ShowResults; end; a Values were varied from 150 to 180 according to brightness of image data. b Values were varied from 5 to 20 according to cell size.

Materials and Methods Fish Red sea bream Pagrus major (700–900 g body weight, 35.0–38.0 cm body length) were obtained from a local supplier and maintained in a 50 L tank for 2 d until killing. The fish were killed by cutting around the connection between the medulla oblongata and the spinal cord, in a procedure called cranial spiking. Two to three fish were used for each experiment.

Microscopic observation of melanophores Spiked fish were soaked and held for 150 h in the experimental solutions listed in Table 1 at 0 °C. Each fish was set on the microscope stage at an appropriate time and the skin was observed under a video microscope OVM 1000N (Olympus, Tokyo, Japan) equipped with a digital image recorder DF-10 (Fujix, Tokyo, Japan). In this study, Cl– was used as a counter ion against any cations instead of other anions to simplify ion compositions for simulating practical usages. Fixed fields were also observed continuously in the system to evaluate choromatosome movements in

Fig. 1 Live specimen of red sea bream

Fig. 2 Red sea bream immediately after death (A) and after storage for 30 min, (B) and 80 h (C) at 0 °C in 10 K + solution

Fig. 3 Chromatosomes in red sea bream skin. M and E indicate melanophore and erythrophore, respectively. The bar represents 50 µm

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Fig. 4 Melanophores (A) before and (B) after image analysis. The bar indicates 50 µm

Fig. 6 Red sea breams soaked in solutions containing various concentrations of K + for 80 h at 0 °C. (A) 10 K + ; (B) 50 K + ; (C) 100 K + ; (D) 300 K + ; (E) 480 K +

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Statistical analysis Image data obtained from five to ten microscopic fields for three individuals were processed as described above, except that one set of field data for each fish were acquired in the fixed field observation. The resulting dispersion indeces were averaged and analysed in a Student’s t-test.

0.5 0.45 0.4 Dispersion index

using a macro program listed in Table 2 and available on the public domain NIH Image 1.57 (developed at the U.S. National Institutes of Health and available from the Internet by anonymous FTP from zippy. nimh.nih.gov). The dispersion index was defined as the ratio of total pixels in a 480 3 640 field to pixels occupied by melanophores, which was used for the evaluation of the dispersion of chromatosome in melanophores.

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

20

40 Time (h)

80

60

Fig. 5 Changes in dispersion indeces of red sea bream skin soaked in different experimental solution during storage at 0 °C. Data are presented as mean ± S.D. (j) = 10 K + ; (d) = 50 K + ; (s) = 100 K + ; (m) = 300 K + ; (h) = 480 K +

Results of 0.31 immediately after spiking rapidly decreased to 0.08 after 5 min in a 480 K + solution. The 300 K + solution also induced a rapid decrease in the dispersion index value (from 0.25 to 0.17 after 5 min and 0.12 after 30 min). On the other hand, solutions containing less than 100 mmol/L K + failed to reduce the value. When a solution containing only 100 mmol/L KCl was applied, the dispersion index value decreased as rapidly as in the case when the 300 K + solution was used.

Discussion The present study revealed that Na + and K + concentrations in the soaking solution affected melanosome movement, that a K + concentration higher than 300 0.4 0.35 0.3 Dispersion index

The colour of the fish skin is very vivid in the living state (Fig. 1). It is noteworthy that the nose area of the fish expressed a red colour. Immediately after spiking, the skin of the same fish rapidly became dark, in particular around the nose area (Fig. 2A). Although the skin darkening was slightly improved after 30 min when soaked with artificial sea water, the vivid colour of the living state was hardly recovered by 80 h (Fig. 2B and C). Microscopic observation revealed that such skin colour changes in red sea bream were expressed by dispersion of chromatosomes in melanophores and erythrophores (Fig. 3). The cell size of melanophore (about 50 to 80 µm) were much greater than those of erythrophores (about 5 to 20 µm). Figure 4 shows chromatophore photo-images before (A) and after (B) treatment by the NIH image macro program listed in Table 2. Fish skin soaked in solutions containing 470 mmol/L Na + plus 10 mmol/L K + (10 K + ), 430 mmol/L Na + plus 50 mmol/L K + (50 K + ), and 380 mmol/L Na + plus 100 mmol/L K + (100 K + ) at 0 °C exhibited relatively high dispersion index values over 80 h (0.2–0.5), while solutions containing 180 mmol/L Na + plus 300 mmol/L K + (300 K + ) and 480 mmol/L K + (480 K + ) prevented chromatophore dispersion over 80 h (Fig. 5). Thus, a rapid decrease in dispersion index of fish skin in 300 K + and 480 K + solutions were achieved within 1 h after spiking of fish. These changes in dispersion index values were consistent with colour changes under macroscopic observation, as shown in Fig. 6. Fish skin expressed a blackish colour, in particular around the nose area, in treatments with 10 K + , 50 K + , and 100 K + solutions, while 300 K + and 480 K + solutions prevented skin darkening over 80 h. However, the 480 K + solution, which reduced the dispersion index most rapidly, caused a weakened red colour. Typical data for each individual are shown in Fig. 7 for the fixed field observation during the initial 60 min immediately after spiking. The dispersion index value

0.25 0.2 0.15 0.1 0.05 0

10

20

30 Time (min)

40

50

60

Fig. 7 Relatively rapid changes in dispersion indices of red sea bream skin during storage in solutions containing various concentrations of K + and a solution containing only 100 mmol/L KCl at 20 °C. The presented typical data were consistent with those in the other experiments. (j) = 10 K + ; (d) = 50 K + ; (s) = 100 K + ; (m) = 300 K + ; (n) = 480 K + ; (h) = 100 mmol/L KCl

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mmol/L prevented skin darkening of red sea bream, and that such movements were almost complete within 10 min of soaking in the solutions. These solutions were effective for 30 min after death of the fish. Therefore, K + solution treatment immediately after death is thought useful to improve skin colour of red sea bream. Because the cell sizes of melanophores were much greater than those of erythrophores, it is believed that the movement of chromatosome in the melanophores plays a more important role in the drastic colour change observed immediately after spiking of fish. We therefore focused on the chromatophore movements of melanophores in the present study. The 300 K + solution induced aggregation of the melanosomes and left the erythrosomes in a dispersed state, resulting in the expression of the most vivid colour of red sea bream skin in the present study. On the other hand, loss of red colour of fish soaked in the 480 K + solution was probably due to the effect of the solution on erythrosome movement. Karlsson et al. (22) demonstrated that squirrel fish erythrophores themselves had a K + -sensitive mechanism for aggregation, which was different from melanophores in previous reports (12–16). Martensson ˚ et al. (23) compared the α-adrenoceptor systems between erythrophores and melanophores of cuckoo wrasse Labrus ossifagus and found differences in the α2-adrenoceptor systems regulating pigment migration between erythrophores and melanophores. A similar variation in the α-adrenoceptor systems might also explain the different movements of melanosomes and erythrosomes in red sea bream in the present study. We are now trying to determine physiological differences between the melanophores and the erythrophores of red sea bream by pharmacological approaches. Although we used solutions with the same osmolarity as sea water to make the cells of fish skin physiological in this study, treatment with only 100 mmol/L KCl solution also had similar effect to that of the 300 K + solution. This is probably due that the balance between Na + and K + being disturbed by the treatment with the solution containing only KCl, inducing influx of K + and membrane depolarization. Further investigations are required to confirm this speculation. Thus, soaking or spraying with KCl aqueous solution may be practical for commercial use. However, because KCl solution had a very strong effect, use of the solution should be limited to a concentration lower than 100 mmol/L. Higher concentration of KCl will not only prevent skin colour darkening but also reduce the valuable red colour.

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References

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